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研究生:施姍汶
研究生(外文):Shan-Wen Shih
論文名稱:海帶多醣水解液乳酸發酵產物之生理活性探討
論文名稱(外文):Studies on Biological Activities of Lactic Acid Fermented Laminaria japonica Polysaccharide-Hydrolysates Product
指導教授:潘崇良
指導教授(外文):Chorng-Liang Pan
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:99
中文關鍵詞:海帶乳酸菌抗氧化抗致突變性
外文關鍵詞:kelplactic acid bacteriaantioxidant propertiesantimutagenicity
相關次數:
  • 被引用被引用:9
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摘 要

本研究探討將海帶 [Laminaria (Lam.) japonica] 多醣萃取液以酵素水解後經乳酸菌發酵之製程,並測試該製品之抗氧化和抗致突變性生理活性。海帶熱水萃取液 (Lam) 經 5 units/mL Agarases 和 Cellulase 於 40oC 降解 12 小時後可得海帶水解液 (L-Lam),由膠體過濾層析測試結果可知其具適合乳酸發酵的寡醣與單醣組成,且主要組成物之分子量介於 153.77-936.80 Da。
以 (A) Lactobacillus (Lb.) rhamnosus BCRC 14068 和Enterococcus (Ent.) faecalis BCRC 13076、(B) Lb. plantarum BCRC 10069 和 Lb. plantarum BCRC 12250、與 (C) Lb. plantarum BCRC 10069 和 Lactococcus (Lc.) lactis BCRC 12315 三組混合菌酛 (接種量為 3% 或 5%, v/v) 各別於 37oC 下發酵2% (w/v) (I) Lam、(II) L-Lam、(III) L-Lam + YE [L-Lam + 0.5% (w/v) yeast extract]、或 (IV) L-Lam + P [L-Lam + 0.5% (w/v) peptone],即可得海帶多醣乳酸發酵產物。B 組乳酸菌酛發酵 (III) L-Lam + YE 4 小時後可達到發酵終點 (pH < 4.6),該組產物之可滴定酸度與乳酸菌數皆為各試驗組中之最高者。
在螯合亞鐵離子能力上,以 C 組菌酛發酵 (IV) L-Lam + P 所得產物之螯合能力為 76.15% 以上。在清除 DPPH 自由基方面,添加氮源發酵組產物 [(III) 和 (IV)] 較未添加氮源之發酵產物 [(I) 和 (II)] 具較高抗氧化性。在 TEAC 方面,各試驗組間亦以 (IV) L-Lam + P 發酵產物呈現較高的抗氧化性 (74.26%-78.56%)。
未經發酵之 (II) L-Lam 對於直接型致突變劑 4NQO 所誘導 Sal. typhimurium TA100 突變之抑制率為 46.2%,雖高於各發酵組產物之突變抑制率,但僅與 A 組菌酛發酵 (III) L-Lam + YE 之突變抑制率有顯著差異 (19.8%)。而未經發酵之 L-Lam 對於間接型致突變劑 B[a]P 所誘導 Sal. typhimurium TA100 突變之抑制率則為 36.9%,除與 C 組發酵 (III) L-Lam + YE 之突變抑制率有顯著差異 (12.6%) 外,與其他各實驗組之海帶發酵產物間則未呈現顯著差異。
以上述 3 組菌酛組合發酵 (II) L-Lam 或 (III) L-Lam + YE 至 pH = 4.4-4.6 後,共可得 6 組海帶發酵產物。於 4oC 下儲藏之期間,6 組發酵產物之 pH 值會隨著儲藏時間的增加而持續下降,3 組菌酛發酵 (III) L-Lam + YE 產物乳酸菌數下降 2.2%-3.4%,且可維持在 8.7 log CFU/mL。
Abstract

This study is to develop the production protocol of using enzyme digested polysaccharide extracts of Laminaria (Lam.) japonica fermented by lactic acid bacteria. And their antioxidant properties and antimutagenicity were examined. The polysaccharide extracts of Lam. japonica which were digested by 5 units/mL cellulase and agarases at 40oC for 12 hrs obtained L-Lam. Data collected from gel permeation chromatography of the L-Lam indicated that it contain oligosaccharides and monosaccharides suitable for lactic fermentation, and the molecular weight of these oligosaccharide majorly between 153.77-936.80 Da.
2% (w/v) (I) Lam, (II) L-Lam, (III) L-Lam-YE [L-Lam + 0.5% (w/v) yeast extract], or (IV) L-Lam-P [L-Lam + 0.5% peptone] was fermented individually with (A) Lactobacillus (Lb.) rhamnosus BCRC 14068 and Enterococcus (Ent.) faecalis BCRC 13076, (B) Lb. plantarum BCRC 10069 and Lb. plantarum BCRC 12250, or (C) Lb. plantarum BCRC 10069 and Lactococcus (Lc.) lactis BCRC 12315 three groups of lactic starters 3% or 5%, (v/v) with inoculum and incubated at 37oC to obtain fermented L-Lam products. (III) L-Lam-YE fermented by group (B) lactic starters could reach the end point of fermentation (pH < 4.6) in 4 hrs, and its titratable acidity value and lactic acid bacteria counts are the highest among the tested groups.
The ability of chelating on Fe2+ ion of group (C) lactic starters fermented (IV) L-Lam-P exhibited over 76.15%. DPPH radical scavenging effect of lactic starters fermented nitrogen source added L-Lam, (III) and (IV), were stronger than the groups without nitrogen source added L-Lam, (I) and (II), fermented by lactic starters did. Lactic starters fermented products devived from (IV), 0.5% peptone added L-Lam, also performed higher antioxidative capacity on TEAC (74.26%-78.56%) that is the best of all fermented products.
The inhibition effect of (II) L-Lam against the mutagenicity induced by direct-acting mutagen 4NQO evaluated by Sal. typhimurium TA100 was 46.2%. It was only significantly and higher than group (A) lactic starters fermented products with (III) L-Lam-YE (19.8%), but it showed no difference to the rest of fermented products. The inhibition effect of L-Lam against the mutagenicity induced by indirect-acting mutagen B[a]P (with S9 mix) and evaluated by Sal. typhimurium TA100 was 36.9%, which was only significantly higher than group (C) lactic starters fermented products with (III) L-Lam-YE (12.6%), but it present no difference to the rest of fermented products.
Three groups of lactic starters individually fermented with (II) L-Lam or (III) L-Lam-YE until pH value falls to 4.4-4.6, then six fermented L-Lam were harvest products. The pH value of these six fermented L-Lam products were declined as storage time increasing. During the storage period that conducted at 4oC for 10 days, the three L-Lam fermented products derived from (III) L-Lam-YE fermented by each of 3 lactic starters showed their lactic acid bacteria counts decreased only 2.2%-3.4%, but still maintained above 8.7 log CFU/mL.
目 錄

目錄 -i
表目錄 -vii
圖目錄 -ix
中文摘要 -xi
英文摘要 -xiii
壹、前言 -1
貳、文獻整理 -3
一、海帶簡介 -3
1. 海帶型態與分佈 -3
2. 海帶成分與利用 -3
二、乳酸菌之簡介 -5
1. 乳酸菌之定義 -5
2. 乳酸菌之抗氧化 -6
3. 乳酸菌之應用與生理功能 -7
A. 調節宿主免疫系統 -7
B. 維持腸道微生物菌相平衡 -8
C. 抗癌機制 -8
D. 提高營養價值、促進維生素的合成及酵素的產生 -9
E. 具抗菌作用 -9
三、海帶之抗氧化特性 -9
1. 自由基與活性氧之形成 -9
2. 氧化傷害與疾病之相關性 -10
3. 抗氧化劑的種類 -12
A. 自由基終止型 -12
B. 還原劑與氧清除劑 -13
C. 單重態氧抑制劑 -13
D. 金屬螯合劑 -13
E. 抗氧化酵素 -14
4. 抗氧化活性測試原理 -14
A. DPPH 自由基清除力指標與其原理 -14
B. 亞鐵離子螯合能力測試指標及其原理 -14
C. Trolox equivalent antioxidant capacity (TEAC) -15
5. 海帶抗氧化成分 -15
四、抗致突變物之作用機制 -16
1. 去致突變物 -17
A. 防止致突變物生成抑制劑 -17
B. 降低致突變物造成 DNA 傷害之阻斷劑 -17
a. 致原致突變之不活化作用 -17
b. 對代謝活化酵素系統之抑制作用 -17
c. 對原致突變物代謝活化物之不活化作用 -18
d. 促進解毒代謝系統 -18
C. 抗氧化劑 -18
2. 生物抗致突變物 -19
A. 作用於 DNA 修補之化合物 19
B. 抑制腫瘤促進與增殖作用之抑制劑 19
3. 抗致突變性之檢測方法 19
A. Histidine requirement 20
B. rfa mutation 21
C. uvrB mutation 21
D. R-factor 21
E. pAQ1 plasmid 21
參、實驗設計 22
肆、材料與方法 23
一、實驗材料 23
1. 原料 23
2. 試驗菌株 23
A. 乳酸菌菌株 23
B. 安氏試驗法 (Ames test) 試驗菌株 23
C. 產洋菜酶海洋菌株 24
3. 藥品 24
4.儀器設備 27
二、實驗方法 27
1. 海帶之ㄧ般成分分析 27
A. 水分含量 28
B. 灰分含量 28
C. 粗蛋白含量 28
D. 粗脂肪含量 28
E. 總醣量的測定 29
2. 乳酸菌菌株之保存與活化 29
A. 乳酸菌菌株之保存 29
B. 乳酸菌菌株之活化 29
3. 產洋菜酶海洋菌株之保存與活化 30
A. 產洋菜酶海洋菌株之保存 30
B. 產洋菜酶海洋菌株之活化 30
4. Agarases 粗酵素液之生產 30
A. Agarases 粗酵素液之生產 30
B. Agarases 活性測定 31
5. 海帶發酵液之製備及分析 32
A. 海帶熱水多醣萃取液之製備 32
B. 以多醣分解酵素降解海帶熱水萃取液 32
C. 還原醣量之測定 33
D. 海帶多醣水溶液及其水解液經 Sephadex 膠體過濾
層析之測定 33
E. 薄層色層分析 (TLC) 33
F. 海帶發酵液之製備 34
a. pH 值之測定 34
b. 可滴定酸度之測定 34
c. 乳酸菌菌數測定 35
d. 總醣量的測定 35
e. 蛋白質含量之測定 35
f. 儲藏試驗 35
6. 海帶乳酸發酵液之抗氧化活性 36
A. 清除 DPPH 自由基能力之測定 36
B. 亞鐵離子螯合能力之測定 36
C. Trolox equivalent antioxidant capacity (TEAC) 36
7. 海帶乳酸發酵液之抗致突變性測定 37
A. Salmonella typhimurium TA100 之保存與活化 37
a. 菌株 TA100 之保存 37
b. 菌株 TA100 之活化 37
B. Salmonella typhimurium TA100 基因型態之確認 38
a. 組胺酸需求性之確認 38
b. rfa 突變之測試 39
c. uvrB 突變之測試 39
d. R-factor 之測試 40
C. 毒性試驗 40
D. 致突變實驗 41
E. 抗致突變性試驗 41
8. 統計與分析 42
伍、結果與討論 43
一、海帶乾燥粉末之一般成分分析 43
二、以四種多醣分解酵素降解海帶熱水萃取液 43
1. 還原醣含量變化 43
2. 膠體過濾層析分析 44
3. 薄層色層分析 45
三、經多醣分解酵素分解之海帶多醣液之發酵實驗結果 46
1. 乳酸菌株組合之選用 46
2. 發酵試驗 47
3. 額外添加氮源之發酵試驗 48
四、海帶乳酸發酵液抗氧化之探討 49
1. 螯合亞鐵離子能力 49
2. 清除 DPPH 自由基能力 49
3. Trolox equivalent antioxidant capacity (TEAC) 50
五、海帶乳酸發酵液抗致突變性之探討 51
1. Salmonella (Sal.) typhimurium TA100 試驗菌株
基因型態之確認 51
A. 組胺酸之需求測定 (Histidine requirement) 51
B. rfa 突變 (rfa mutation) 之測試 51
C. uvrB 突變 (uvrB mutation) 之測試 52
D. R-factor 之測試 52
2. 毒性試驗 52
3. 致突變試驗 53
4. 抗致突變性試驗 53
六、海帶乳酸發酵液儲藏試驗之探討 54
1. pH 值與可滴定酸度含量變化 54
2. 乳酸菌菌數變化 55
陸、結論 57
柒、參考文獻 59

表 目 錄

表一、 海帶的一般成份 71
表二、 3% (v/v) 乳酸菌酛發酵海帶熱水萃取液 (Lam) 及海帶
水解液 (L-Lam) 在 37oC 發酵過程中 pH 值、可滴定
酸度、還原醣含量與好氣性生菌數及乳酸菌菌數變化 81
表三、 5% (v/v) 乳酸菌酛發酵海帶熱水萃取液 (Lam) 及海帶
水解液 (L-Lam) 在 37oC 發酵過程中 pH 值、可滴定
酸度、還原醣含量與好氣性生菌數及乳酸菌菌數變化 82
表四、 3% (v/v) 乳酸菌酛發酵 L-Lam + 0.5% yeast extract 在
37oC 發酵過程中 pH 值、可滴定酸度、還原醣含量、
總醣、蛋白質含量與好氣性生菌數及乳酸菌菌數變化 83
表五、 5% (v/v) 乳酸菌酛發酵 L-Lam + 0.5% yeast extract 在
37oC 發酵過程中 pH 值、可滴定酸度、還原醣含量、
總醣、蛋白質含量與好氣性生菌數及乳酸菌菌數變化 84
表六、 3% (v/v) 乳酸菌酛發酵 L-Lam + 0.5% peptone 在 37oC
發酵過程中 pH 值、可滴定酸度、還原醣含量、總醣、
蛋白質含量與好氣性生菌數及乳酸菌菌數變化 85
表七、 5% (v/v) 乳酸菌酛發酵 L-Lam + 0.5% peptone 在 37oC
發酵過程中 pH 值、可滴定酸度、還原醣含量、總醣、
蛋白質含量與好氣性生菌數及乳酸菌菌數變化 86
表八、 乳酸菌酛發酵海帶熱水萃取液及海帶水解液對螯合亞鐵
離子能力之測定 87
表九、 乳酸菌酛發酵添加 0.5% (w/v) yeast extract or peptone 之
海帶水解液對螯合亞鐵離子能力之測定 88
表十、 乳酸菌酛發酵海帶熱水萃取液及海帶水解液對 DPPH
自由基之清除效應 89
表十一、乳酸菌酛發酵添加 0.5% (w/v) yeast extract or peptone 之
海帶水解液對 DPPH 自由基之清除效應 90
表十二、乳酸菌酛發酵海帶熱水萃取液及海帶水解液對 TEAC
之測定 91
表十三、乳酸菌酛發酵添加 0.5% (w/v) yeast extract or peptone 之
海帶水解液對 TEAC 之測定 92
表十四、沙門氏菌株變異株 Sal. typhimurium TA 100 之基因
型態檢測 93
表十五、海帶水解液及其乳酸菌發酵產物對 Sal. typhimurium
TA100 之毒性評估 94
表十六、海帶水解液及其乳酸菌發酵產物對 Sal. typhimurium
TA100 之致突變性評估 95
表十七、海帶水解液及其乳酸菌發酵產物對 4NQO 和 B[a]P
之抗致突變性評估 96

圖 目 錄

圖一、 2% (w/v) 海帶熱水萃取液 (Lam) 分別經 1 unit/mL
��-Galactosidase、��-Amylase、Agarases (MAEF108-
agarases 和 MA103-agarases)、或 Cellulase 分解後之
還原醣含量變化 72
圖二、 2% (w/v) 海帶熱水萃取液 (Lam) 分別經 1、2、3、4、
或 5 units/mL Cellulase 分解後之還原醣含量變化 73
圖三、 2% (w/v) 海帶熱水萃取液 (Lam) 分別經 5
units/mL Cellulase、5 units/mL Cellulase + 5 units/mL
Agarases (MAEF108-agarase 和 MA103-agarases)、
或 5 units/mL Agarases (MAEF108-agarase
和 MA103-agarases) 分解後之還原醣含量變化 74
圖四、 2% (w/v) 海帶熱水萃取液 (Lam) 中醣類與蛋白質組成
物之 Sephadex G-10 膠體過濾層析圖 75
圖五、 2% (w/v) 海帶熱水萃取液 (Lam) 先經 5 units/mL
Cellulase 及 5 units/mL Agarases (MAEF108-agarases 和
MA103-agarases) 於 40oC 下分解 12 小時後之水解物
中醣類組成物之 Sephadex G-10 膠體過濾層析圖 76
圖六、 2% (w/v) 海帶熱水萃取液 (Lam) 分別經 1 unit/mL
Cellulase (CE)、Agarases (MAEF108-agarases 和
MA103-agarases) (AG)、β-Galactosidase (GA)、
α-Amylase (AM) 或 5 units/mL Cellulase 及 5 units/mL
Agarases (MAEF108-agarases 和 MA103-agarases)
(MIX) 分解後所得水解液之薄層色層分析圖 77
圖七、 3% (v/v) 乳酸菌酛發酵海帶水解液 (L-Lam) 在 37oC
發酵 12 小時中之 pH 值變化 78
圖八、 3% (v/v) 乳酸菌酛發酵海帶水解液 (L-Lam) 在 37oC
發酵 12 小時中之可滴定酸度變化 79
圖九、 3% (v/v) 乳酸菌酛發酵海帶水解液 (L-Lam) 在 37oC
發酵 12 小時中之還原醣含量變化 80
圖十、 乳酸菌酛發酵海帶水解液於 4oC 儲藏期間之 pH 值
變化 97
圖十一、乳酸菌酛發酵海帶水解液於 4oC 儲藏期間之可滴定
酸度變化 98
圖十二、乳酸菌酛發酵海帶水解液於 4oC 儲藏期間之乳酸菌
菌數變化 99
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